1. Field of the Invention
The present invention relates to a new product consisting of a protein. It also relates to a process for isolating such a protein. Another object of the invention is the use of this new product in a therapeutic composition and in particular an immunoregulating and anti-allergic therapeutic composition. In one of its aspects, the invention therefore pertains to the immunological and allergic field.
2. Description of the Prior Art
It is known that allergic manifestations are very frequent and are due to a large number of factors. Allergic illnesses are tenacious owing to the very reason of the constant or occasional presence of factors producing the allergic reactions. It is therefore desirable to increase the immunizing reactions of the organism to improve its resistance to exterior attacks and in particular the factors responsible for the allergic reactions.
Among the causes of allergy plant pollens are often mentioned. One skilled in the art may in this respect refer to the very documentated article of Raymonde BARREAU and Jean-Rene MALLET in the "Journal de Medecine de Bordeaux" 1958, 135, 1379-1389. This bibliographical reference refers first of all to the social importance of allergic diseases and in particular those due to pollen. Many plants exist the blossoming of which, accompanied by the production of pollen, produces allergic reactions for relatively long periods of the year. The article in question and the publications mentioned at the end of the latter are inserted by way of reference in the present disclosure.
An object of the present invention is to provide a protein possessing particular immunological properties. This protein is of use in combating allergic diseases and manifestations.
Thus the invention relates to a new product consisting of a protein having a molecular weight of about 14 000 and isoelectric point of about 4.6, its absorption spectrum having an absorption of .epsilon.=0.12 units of optical density O.D., at 280 nm for an aqueous solution of 1 mg (dry weight) per ml of water.
Another object of the invention is to provide a process for isolating said protein from the pollen of a graminaceous plant, namely orchard grass.
Thus the invention has for further object to provide a process for obtaining a new protein product comprising putting the pollen, especially of graminaceous plant and preferably of orchard grass or Dactylis glomerata in contact with an aqueous medium, collecting the supernatent part, subjecting the soluble extract to a fractionating treatment and isolating the protein having a molar mass of about 14,000 g (molecular weight: 14,000 daltons) and an isoelectric point of about 4.6.
The starting plant material in the process of the invention is preferably a graminaceous plant termed Dactylis glomerata. The pollen of this plant is put in contact with an aqueous medium so as to provide a soluble fraction. The simplest manner of proceeding consists in placing the pollen in water and allowing it to incubate until the soluble fraction is extracted. It is advantageous to work at room temperature. The incubation period depends on the volume of the reaction medium employed. In practice, at room temperature, incubation periods of the order of one hour have been found suitable. The relative amounts of aqueous medium, in particular distilled water and orchard grass pollen, may vary within wide ranges. The weight of pollen in proportion to the volume of water may be from 10 to 200 parts by weight, for example of the order of 100 parts by weight per volume of water.
At the end of the incubation, the supernatent part is separated from the solid parts still present in the reaction medium and the soluble fraction is then fractionated so as to isolate the protein having the previously defined characteristics.
For the characterization of the new protein, there may be employed the isoelectrofocusing preparing technique described by RADOLA, B. J. (1973), Biochim. Biophys. Acta, 295, 412-428. This technique is known to one skilled in the art who may, if necessary, refer to this last bibliographical reference for the details of procedure. The fractionating method in question permits, by varying the pH, collecting the purified fractions in a thin layer of granulous gel (for example Sephadex or Biorad). When the soluble fraction extracted from the orchard grass pollen is treated with the considered technique, more than 60 bands are found to be present between pH 3.8 and 9. The protein according to the invention is separated at pH 4.6.
In an alternative embodiment, the new protein may be isolated by the chromatofocusing technique, such as that for example described in the printed article named "Chromatofocusing" distributed by Pharmacia Fine Chemicals AB Box 175 Uppsala Sweden; see also "Chromatofocusing isoelectric focusing on ion exchange columns", page 17-44 J. Chromatogr. 150 (1978).
The protein of the invention has particular immunological properties owing to its immunoregulating activity. Indeed, instead of inducing the synthesis of the homocytotropic IgE and IgG (for the definition of this expression see for example page 1010 of the work "Immunologie" by Paul Bordet (1972), Ed. Flammarion), specific immunoglobulins of the allergenic response, it strongly inhibits the synthesis of this class of immunoglobulins and only slightly diminishes that of the IgA, IgM and other IgGs. Investigations have shown in particular that the new protein is capable of inhibiting the synthesis of homocytotropic IgE and IgG after sensitization of a receiving organism with orchard grass pollen. These results show the specific activity of the immunoregulating protein according to the invention.
The protein according to the invention is therefore of utility in a therapeutic composition for allergic diseases and in particular pollinosis. Thus the invention concerns a therapeutic composition containing the protein defined hereinbefore as the active agent. More particularly, the invention concerns an immunoregulating therapeutic composition and an anti-allergic therapeutic composition containing said protein as the active agent.
Another object of the invention is to provide pharmaceutical compositions containing an effective quantity of the new protein in association with a pharmaceutically-acceptable vehicle suitable for the considered administration. For the administration, various routes, in particular, the oral, or parenteral route by inhalation or by injection are possible. It will be clear that the vehicles must be chosen differently in each of these routes. The galenical forms may be easily determined by one skilled in the art in accordance with the chosen mode of administration.
The new protein of the invention is very soluble in water. 5 mg of said protein are easily dissolved in 1 ml of water. It can be freeze-dried. The freeze-dried powder preserves all of the properties of the ordinary powder.
One skilled in the art will therefore understand that the product of the invention may be employed in very varied pharmaceutical compositions. Without the following indications having a limiting character, the composition may be in the form of injectable solutions. The product may also be in the form of a dry or freeze-dried powder, for example for the preparations suitable for inhalation, in particular in the form of an aerosol. The solid product may also be adsorbed on an adjuvant and be thus employed in an injection. Aqueous or oily compositions may also be envisaged for administrations by nasal/ophthalmic or oral instillation, or by inhalation. Other traditional galenical forms for oral administration may also be suitable, such as gelules, capsules, tablets, and like forms.
The therapeutic properties of the new protein act at small doses and no undesirable secondary effect has been found.
It will be observed that, for the envisaged applications, the protein of the invention has a group of very advantageous properties. It is the result of the fractionation of a product of natural origin, namely the pollen of a graminaceous plant. It is active at a low dose. Its toxicity is nonexistent in the normal conditions of application. Thus, when injected at the dose of 100 .mu.g per mouse, namely 5 mg/kg by weight in man, no toxicity was found, whereas the most effective dose is at values which are 1/1000th of this value (0.1 .mu.g instead of 100 .mu.g in the mouse). This last property is very important, since the dangers of the use of unmodified allergens, for example of a total extract of pollen, are known. Very serious and even mortal accidents have occurred in the past when such products were injected in subjects suffering from allergy within the framework of a densitization treatment.
It is also of interest that the protein have a selective effect in the inhibition of antibodies formed by allergic reactions. The preferred field of utilization is that of the combatting of allergic reactions produced by the pollens of graminaceous plants.
In the adult man, a suitable posology for the treatment of pollinosis may range from 1 .mu.g to 5 mg and is preferably of the order of 0.01 mg to be taken in a single injection.
By the oral route, doses of up to 10 mg are possible. For local application, the allowed doses may be as much as 100 mg.
The invention will now be illustrated, without in any way limiting the scope thereof, by the following description which gives an example of the isolation of the new protein from the pollen of orchard grass and the results of some pharmacological investigations.